Alkaline stable fc-binding proteins for affinity chromatography

ABSTRACT

The present invention relates to Fc binding proteins comprising one or more Fc binding domains wherein at least one domain comprises of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6 or 21. The invention further relates to affinity matrices comprising the Fc binding proteins of the invention. The invention also relates to a use of these Fc binding proteins or affinity matrices for affinity purification of immunoglobulins and to methods of affinity purification using the Fc binding proteins of the invention.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of, and claims the benefit ofpriority to, U.S. patent application Ser. No. 16/324,842, filed Feb. 11,2019, entitled “Alkaline Stable FC-Binding Proteins for AffinityChromatography,” which is a 371 Application of PCT/EP2017/069979 filedAug. 6, 2017, entitled: “Alkaline Stable FC-Binding Proteins forAffinity Chromatography,” which claims priority to European PatentApplication No. 16205707.9, filed Dec. 21, 2016, entitled“Immunoglobulin-Binding Protein Variants with Improved AlkalineStability,” and European Patent Application No. 16183710.9, filed Aug.11, 2016, entitled “Immunoglobulin-Binding Protein Variants withImproved Alkaline Stability,” which application is incorporated hereinby reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Feb. 28, 2019, is named1580_00028_SL.txt and is 24,097 bytes in size.

FIELD OF THE INVENTION

The present invention relates to Fc binding proteins comprising one ormore Fc binding domains wherein at least one domain comprises of anamino acid sequence selected from the group consisting of SEQ ID NOs:1-6 or 21. The invention further relates to affinity matrices comprisingthe Fc binding proteins of the invention. The invention also relates toa use of these Fc binding proteins or affinity matrices for affinitypurification of immunoglobulins and to methods of affinity purificationusing the Fc binding proteins of the invention.

BACKGROUND OF THE INVENTION

Many biotechnological and pharmaceutical applications require theremoval of contaminants from a sample containing antibodies. Anestablished procedure for capturing and purifying antibodies is affinitychromatography using the bacterial cell surface Protein A fromStaphylococcus aureus as selective ligand for immunoglobulins (see, forexample, review by Huse et al., J. Biochem. Biophys. Methods 51, 2002:217-231). Wild-type Protein A binds to the Fc region of IgG moleculeswith high affinity and selectivity and is stable at high temperaturesand in a wide range of pH values. Variants of Protein A with improvedproperties such as alkaline stability are available for purifyingantibodies and various chromatographic matrices comprising Protein Aligands are commercially available. However, in particular wild-typeProtein A based chromatography matrices show a loss of binding capacityfor immunoglobulins following exposure to alkaline conditions.

Technical Problems Underlying the Present Invention

Most large scale production processes for antibodies or Fc-containingfusion proteins use Protein A for affinity purification. However, due tolimitations of Protein A applications in affinity chromatography thereis a need in the art to provide novel Fc binding proteins with improvedproperties that specifically bind to immunoglobulins in order tofacilitate affinity purification of immunoglobulins. To maximallyexploit the value of the chromatographic matrices comprising Fc bindingproteins it is desirable to use the affinity ligand matrices multipletimes. Between chromatography cycles, a thorough cleaning procedure isrequired for sanitization and removal of residual contaminants on thematrix. In this procedure, it is general practice to apply alkalinesolutions with high concentrations of NaOH to the affinity ligandmatrices. Wild-type Protein A domains cannot withstand such harshalkaline conditions for an extended time and quickly lose bindingcapacity for immunoglobulin. Accordingly, there is an ongoing need inthis field to obtain novel alkaline-stable proteins capable of bindingimmunoglobulins.

The present invention provides alkaline stable immunoglobulin bindingproteins that are particularly well-suited for affinity purification ofimmunoglobulins but overcome the disadvantages of the prior art. Inparticular, a significant advantage of the alkaline stable Fc bindingproteins of the invention is their improved stability at high pH, forexample compared to wild type protein A or to a parental protein.

The above overview does not necessarily describe all problems solved bythe present invention.

SUMMARY OF THE INVENTION

A first aspect of the present invention is to provide a Fc bindingprotein suitable for affinity purification. This is achieved with thealkaline stable immunoglobulin (Ig) binding protein comprising one ormore Fc binding domains, wherein at least one Fc binding domaincomprises, essentially consists, or consists of an amino acid sequenceof SEQ ID NOs: 1-6 or 21. In one embodiment the Fc binding proteincomprises of 2, 3, 4, 5, or 6 Fc binding domains as defined above linkedto each other. In some embodiments, the linker connecting the domains isa peptide linker. In preferred embodiments, the Fc binding protein isconjugated to a solid support.

In some embodiments, the protein is a homo-multimer, while in someembodiments, the protein is a hetero-multimer.

In some embodiments, at least one of the domain of the protein is aderivative of any one of SEQ ID NOs 1-6 or 21, wherein the derivativehas an amino acid sequence that is 100% identical to one of SEQ ID NOs:1-6 or 21 except that it has a deletion of 1, 2, 3, or 4 amino acidswithin the first 4 amino acids of its N-terminus (position 1, 2, 3,and/or 4) and/or a deletion of 1 or 2 amino acids at the C-terminus(positon 57 and/or 58) relative to the one of SEQ ID NOs:1-6 or 21 uponwhich is based.

In some embodiments, the protein has less than a 15% reduction inbinding capacity following an incubation in 0.5 M NaOH for at least 5hours. For example, the protein may have less than a 10% or less than a5% reduction in binding capacity following an incubation in 0.5 M NaOHfor 6 hours.

In a second aspect, the present invention relates to an affinityseparation matrix comprising the Fc binding protein of the first aspect.

In a third aspect, the present invention relates to a use of the Fcbinding protein of the first aspect or of the affinity separation matrixof the second aspect for affinity purification of immunoglobulins orproteins comprising a Fc sequence of immunoglobulins.

In a fourth aspect, the present invention relates to a method ofaffinity purification of immunoglobulins or proteins comprising a Fcsequence of immunoglobulins comprising the steps of (a) providing aliquid containing an immunoglobulin; (b) providing an affinityseparation matrix comprising an immobilized Fc binding protein of thefirst aspect coupled to said affinity separation matrix; (c) contactingsaid liquid and said affinity separation matrix, wherein saidimmunoglobulin binds to said immobilized Fc binding protein; and (d)eluting said immunoglobulin from said matrix, thereby obtaining aneluate containing said immunoglobulin. In some embodiments, washingsteps can be introduced between steps (c) and (d) of the disclosedmethod. In some embodiments of the disclosed uses and methods, there isgreater than or equal to 95% elution of the protein comprising a Fcsequence at a pH of 3.5 or higher. For example, there is greater than orequal to 98% elution of the protein comprising a Fc sequence at a pH of3.5 or higher.

In another aspect, the present invention relates to a method of affinitypurification of a protein comprising an Fc sequence, the methodcomprising: (a) contacting an affinity separation matrix comprising atleast one Fc binding protein of the first aspect coupled thereto with asolution containing a protein comprising an Fc sequence under conditionsthat permit binding of said at least one Fc binding protein to saidprotein comprising an Fc sequence; and (b) eluting the bound proteincomprising an Fc sequence from said affinity purification matrix.

This summary of the invention does not necessarily describe all featuresof the present invention. Other embodiments will become apparent from areview of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Analysis of the alkaline stability of different Fc bindingdomains immobilized on Sepharose 6B matrix after 6 h 0.5 M NaOHtreatment. Fc binding domains cs24 (SEQ ID NO: 1) and cs26 (SEQ ID NO:2) show significantly improved stability at high pH compared to parentaldomain IB24 (SEQ ID NO: 17).

FIG. 2. Analysis of the activity of Fc binding domains immobilized onPraesto™ Pure 45 matrix pH 9.5 after incubation for 6 h at 0.5 M NaOH.Fc binding domains cs24 (SEQ ID NO: 1), cs24a (SEQ ID NO: 3), cs24b (SEQID NO: 5), cs26 (SEQ ID NO: 2), cs26a (SEQ ID NO: 4), and cs26b (SEQ IDNO: 6).

FIG. 3. Analysis of the activity of Fc binding domains immobilized onPraesto™ Pure 85 matrix (panel A) and on Praesto™ Pure 45 matrix (panelB) at pH 9.5 after incubation for 6 h, and 24 h (panel A), and 6 h, 24h, and 36 h (panel B) at 0.5 M NaOH. Fc binding domains cs24 (SEQ ID NO:1), cs24a (SEQ ID NO: 3), cs24b (SEQ ID NO: 5), cs26 (SEQ ID NO: 2),cs26a (SEQ ID NO: 4), and cs26b (SEQ ID NO: 6), compared to wildtypedomain C.

FIG. 4. Analysis of elution of polyclonal hlgG from Fc binding domainscs24 (SEQ ID NO: 1), cs24a (SEQ ID NO: 3), cs24b (SEQ ID NO: 5), cs26(SEQ ID NO: 2), cs26a (SEQ ID NO: 4), and cs26b (SEQ ID NO: 6) at pH 3.5and 2.0. Panel A shows a representative elution test. Step yield at 3.5pH elution for all Fc domains was greater than 98% (panel B), farexceeding the elution of Protein A domain C.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

Preferably, the terms used herein are consistent with the definitionsprovided in “A multilingual glossary of biotechnological terms: (1UPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kolbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps.

As used in the description of the invention and the appended claims, thesingular forms “a”, “an” and “the” are used interchangeably and intendedto include the plural forms as well and fall within each meaning, unlessthe context clearly indicates otherwise. Also, as used herein, “and/or”refers to and encompasses any and all possible combinations of one ormore of the listed items, as well as the lack of combinations wheninterpreted in the alternative (“or”).

The term “about”, as used herein, encompasses the explicitly recitedamounts as well as deviations therefrom of ±10%. More preferably, adeviation 5% is encompassed by the term “about”.

Several documents (for example: patents, patent applications, scientificpublications, manufacturer's specifications, instructions, GenBankAccession Number sequence submissions etc.) are cited throughout thetext of this specification. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention. Some of the documents cited herein arecharacterized as being “incorporated by reference”. In the event of aconflict between the definitions or teachings of such incorporatedreferences and definitions or teachings recited in the presentspecification, the text of the present specification takes precedence.

All sequences referred to herein are disclosed in the attached sequencelisting that, with its whole content and disclosure, is a part of thisspecification.

In the context of the present invention, the term“immunoglobulin-binding protein” is used to describe proteins that arecapable to specifically bind to the Fc region of an immunoglobulin. Dueto this specific binding to the Fc region, the immunoglobulin-bindingproteins of the invention are capable of binding to entireimmunoglobulins, to immunoglobulin fragments comprising the Fc region,to fusion proteins comprising a Fc region of an immunoglobulin, and toconjugates comprising a Fc region of an immunoglobulin. While the“immunoglobulin-binding proteins” of the invention herein exhibitspecific binding to the Fc region of an immunoglobulin, it is notexcluded that “immunoglobulin-binding proteins” can additionally bindwith reduced affinity to other regions, such as Fab regions ofimmunoglobulins.

Throughout this specification, the term “immunoglobulin-binding protein”is often abbreviated as “Fc binding protein” or “Fc-binding protein”.

In preferred embodiments of the present invention, the Fc bindingprotein comprises one or more Fc binding domains.

The term “dissociation constant” or “KD” defines the specific bindingaffinity. As used herein, the term “KD” (usually measured in “mol/L”,sometimes abbreviated as “M”) is intended to refer to the dissociationequilibrium constant of the particular interaction between a firstprotein and a second protein. In the context of the present invention,the term KD is particularly used to describe the binding affinitybetween an immunoglobulin-binding protein and an immunoglobulin.

A protein of the invention is considered to bind to an immunoglobulin ifit has a dissociation constant KD to immunoglobulin of at least 1 μM orless, or preferably 100 nM or less, more preferably 50 nM or less, evenmore preferably 10 nM or less. For instance, all of the Fc bindingdomains disclosed in SEQ ID Nos: 1-6 and 21 bind lgG1 with a KD of lessthan 1 μM or less.

The term “binding” according to the invention preferably relates to aspecific binding. “Specific binding” means that a Fc binding protein ofthe invention binds stronger to an immunoglobulin (or Fc sequence of animmunoglobulin) for which it is specific compared to the binding toanother non-immunoglobulin target.

The immunoglobulin as understood herein can include, but is notnecessarily limited to, mammalian IgG, such as human IgG-i, human lgG2,human lgG4, mouse IgG-i, mouse lgG2A, mouse lgG2 IgGi, rat lgG2C, goatIgd, goat lgG2, bovine lgG2, guinea pig IgG, rabbit IgG; human IgM,human IgA; and immunoglobulin fragments comprising a Fc region, fusionproteins comprising a Fc region of an immunoglobulin, and conjugatescomprising a Fc region of an immunoglobulin. Notably, naturallyoccurring protein A domains and artificial Fc binding proteins of theinvention do not bind to human lgG3.

The terms “protein” and “polypeptide” refer to any linear molecularchain of two or more amino acids linked by peptide bonds and does notrefer to a specific length of the product. Thus, “peptides”, “protein”,“amino acid chain,” or any other term used to refer to a chain of two ormore amino acids, are included within the definition of “polypeptide,”and the term “polypeptide” may be used instead of, or interchangeablywith any of these terms. The term “polypeptide” is also intended torefer to the products of post-translational modifications of thepolypeptide, including without limitation glycosylation, acetylation,phosphorylation, amidation, proteolytic cleavage, modification bynon-naturally occurring amino acids and similar modifications which arewell-known in the art. Thus, Fc binding proteins comprising two or moreprotein domains also fall under the definition of the term “protein” or“polypeptides”.

The term “alkaline stable” or “alkaline stability” or “caustic stable”or “caustic stability” (abbreviated as “cs” herein) refers to theability of the Fc binding protein of the invention to withstand alkalineconditions without significantly losing the ability to bind toimmunoglobulins. The skilled person in this field can easily testalkaline stability by incubating a Fc binding protein with sodiumhydroxide solutions, e.g., as described in the Examples, and subsequenttesting of the binding activity to immunoglobulin by routine experimentsknown to someone skilled in the art, for example, by chromatographicapproaches.

Fc binding proteins of the invention as well as matrices comprising Fcbinding proteins of the invention exhibit an “increased” or “improved”alkaline stability, meaning that the molecules and matricesincorporating said Fc binding proteins are stable under alkalineconditions for an extended period of time relative to the parental Fcbinding protein, i.e. do not lose the ability to bind to immunoglobulinsor lose the ability to bind to immunoglobulins to a lesser extent thanthe parental Fc binding protein.

The terms “binding activity” refer to the ability of a Fc bindingprotein of the invention to bind to immunoglobulin. For example, thebinding activity can be determined before and/or after alkalinetreatment. The binding activity can be determined for a Fc bindingprotein or for a Fc binding protein coupled to a matrix, i.e. for animmobilized binding protein. The term “artificial” refers to an objectthat is not naturally occurring, i.e. the term refers to an object thathas been produced or modified by man. For example, a polypeptide orpolynucleotide sequence that has been generated by man (e.g., forexample in a laboratory by genetic engineering, by shuffling methods, orby chemical reactions, etc.) or intentionally modified is artificial.

The term “parental” in the term “parental Fc binding protein” or“parental Fc binding domain” as used herein refers to a Fc bindingprotein that is subsequently modified to generate a variant of saidparental protein or domain. Such parent proteins or domains may be anartificial Fc binding domain, as disclosed herein as IB24 (SEQ ID NO:17) or IB26 (SEQ ID NO: 18).

The term “conjugate” as used herein relates to a molecule comprising oressentially consisting of at least a first protein attached chemicallyto other substances such as to a second protein or a non-proteinaceousmoiety.

The term “substitution” or “amino acid substitution” refers to anexchange of an amino acid at a particular position in a parentpolypeptide sequence by another amino acid. Given the known geneticcode, and recombinant and synthetic DNA techniques, the skilledscientist can readily construct DNAs encoding the amino acid variants.

The term “amino acid sequence identity” refers to a quantitativecomparison of the identity (or differences) of the amino acid sequencesof two or more proteins. “Percent (%) amino acid sequence identity” withrespect to a reference polypeptide sequence is defined as the percentageof amino acid residues in a sequence that are identical with the aminoacid residues in the reference polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity.

To determine the sequence identity, the sequence of a query protein isaligned to the sequence of a reference protein. Methods for alignmentare well-known in the art.

The term “fused” means that the components are linked by peptide bonds,either directly or via peptide linkers.

The term “fusion protein” relates to a protein comprising at least afirst protein joined genetically to at least a second protein. A fusionprotein is created through joining of two or more genes that originallycoded for separate proteins. Thus, a fusion protein may comprise amultimer of identical or different proteins which are expressed as asingle, linear polypeptide

As used herein, the term “linker” refers in its broadest meaning to amolecule that covalently joins at least two other molecules. In typicalembodiments of the present invention, a “linker” is to be understood asa moiety that connects a Fc binding domain with at least one further Fcbinding domain, i.e. a moiety linking two protein domains to each otherto generate a multimer. In preferred embodiments, the “linker” is apeptide linker, i.e. the moiety linking the two protein domains is onesingle amino acid or a peptide comprising two or more amino acids.

The term “chromatography” refers to separation technologies which employa mobile phase and a stationary phase to separate one type of molecules(e.g., immunoglobulins) from other molecules (e.g., contaminants) in thesample. The liquid mobile phase contains a mixture of molecules andtransports these across or through a stationary phase (such as a solidmatrix). Due to the differential interaction of the different moleculesin the mobile phase with the stationary phase, molecules in the mobilephase can be separated.

The term “affinity chromatography” refers to a specific mode ofchromatography in which a ligand coupled to a stationary phase interactswith a molecule (i.e. immunoglobulin) in the mobile phase (the sample)i.e. the ligand has a specific binding affinity for the molecule to bepurified. As understood in the context of the invention, affinitychromatography involves the addition of a sample containing animmunoglobulin to a stationary phase which comprises a chromatographyligand, such as a Fc binding protein of the invention.

The terms “solid support” or “solid matrix” are used interchangeably forthe stationary phase.

The terms “affinity matrix” or “affinity separation matrix” or “affinitychromatography matrix”, as used interchangeably herein, refer to amatrix, e.g., a chromatographic matrix, onto which an affinity ligande.g., a Fc binding protein of the invention is attached. The ligand(e.g., Fc binding protein) is capable of specific binding to a moleculeof interest (e.g., an immunoglobulin or a Fc-containing protein) whichis to be purified or removed from a mixture.

The term “affinity purification” as used herein refers to a method ofpurifying immunoglobulins or Fc-containing proteins from a liquid bybinding the immunoglobulins or Fc-containing proteins to a Fc bindingprotein that is immobilized to a matrix. Thereby, all other componentsof the mixture except immunoglobulins or Fc-containing proteins areremoved. In a further step, the bound immunoglobulins or Fc-containingproteins can be eluted in purified form.

Embodiments of the Invention

The present invention will now be further described. In the followingpassages different aspects of the invention are defined in more detail.Each aspect defined below may be combined with any other aspect oraspects unless clearly indicated to the contrary. In particular, anyfeature indicated as being preferred or advantageous may be combinedwith any other feature or features indicated as being preferred oradvantageous.

In a first aspect the present invention is directed to a Fc bindingprotein, comprising one or more Fc binding domains, wherein at least oneFc binding domain comprises, consists essentially of, or consists of anamino acid sequence of SEQ ID NOs: 1-6 or 21. One advantage of thedisclosed Fc binding domains and proteins comprising said domains isthat they remain stable even after alkaline treatment, in particular ascompared to parent proteins and other known Fc binding proteins. Forexample, in some embodiments, the disclosed Fc binding domains andproteins comprising said domains may be greater than at least about 15%,at least about 20%, at least about 25%, or at least about 30% morestable than Protein A domain C after being exposed to alkalineconditions. In other words, the disclosed Fc binding domains have lessreduction of binding capacity following >5 hour incubation time with 0.5M NaOH when compared to Protein A domain C. Thus, in some embodiments,the disclosed Fc proteins have less than a 20% reduction in bindingcapacity following an incubation in 0.5 M NaOH for at least 5 hours(e.g., 6 hours). In some embodiments, the reduction in binding capacityof the disclosed Fc proteins following an incubation in 0.5 M NaOH forat least 5 hours may be less than about 15%, less than about 10%, orless than about 5%.

All Fc binding proteins of the invention bind to Immunoglobulin with adissociation constant KD below 1 μM, or preferably below 100 nM, or evenmore preferably 10 nM or less. Methods for determining bindingaffinities of Fc binding proteins or domains, i.e. for determining thedissociation constant KD, are known to a person of ordinary skill in theart and can be selected for instance from the following methods known inthe art: Surface Plasmon Resonance (SPR) based technology, Bio-layerinterferometry (BLI), enzyme-linked immunosorbent assay (ELISA), flowcytometry, isothermal titration calorimetry (ITC), analyticalultracentrifugation, radioimmunoassay (RIA or IRMA) and enhancedchemiluminescence (ECL). Some of the methods are described further inthe Examples. Typically, the dissociation constant KD is determined at20° C., 25° C., or 30° C. If not specifically indicated otherwise, theKD values recited herein are determined at 22° C.+/−3° C. by surfaceplasmon resonance. In an embodiment of the first aspect, the Fc bindingprotein has a dissociation constant KD to human Igd in the range between0.1 nM and 100 nM, preferably between 0.1 nM and 10 nM.

As shown in the examples below, surprisingly and unexpected the Fcbinding proteins of the invention were found to bind to IgG even afterprolonged alkaline treatment. In some embodiments, the Fc bindingproteins of the invention exhibit an improved alkaline stability ascompared to a corresponding parental protein. The alkaline stability ofthe Fc binding protein is determined by comparing the loss inIgG-binding activity of the Fc binding protein after 6 h incubation in0.5 M NaOH, as compared to the loss in IgG-binding activity of thecorresponding parental protein after 6 h incubation in 0.5 M NaOH. Theloss of binding activity is determined by comparing binding activitybefore and after 0.5 M NaOH incubation for 6 hours.

As shown by the comparative data in FIG. 1, the IgG binding activity ofcs24 and cs26 is increased by at least about 30% compared to IB24. Thisis an unexpected and advantageous property of cs24 and cs26 as comparedto parental IB24. FIG. 2 shows that all Fc binding proteins of SEQ IDNOs: 1-6 have at least 87.6% binding activity remaining activity after 6h incubation at 0.5 M NaOH

In one embodiment of the invention, the Fc binding protein comprises 2,3, 4, 5, or 6 Fc binding domains linked to each other, i.e. the Fcbinding protein can be a monomer, dimer, trimer, tetramer, pentamer, orhexamer.

In some embodiments, the domains are selected from the group consistingof SEQ ID NOs: 1-6 and 21. In other embodiments, the domains arederivatives of SEQ ID NOs: 1-6 or 21 and further wherein each derivativehas an amino acid sequence that is 100% identical to one of SEQ ID NOs:1-6 except that it has a deletion of 1, 2, 3, or 4 amino acids withinthe first 4 amino acids of its N-terminus and/or a deletion of 1 or 2amino acids at the C-terminus (position 57 and/or 58) relative to theone of SEQ ID NOs:1-6 or 21 upon which it is based (see, for example,SEQ ID NOs: 7-16).

Multimers of the invention are fusion proteins generated artificially,generally by recombinant DNA technology well-known to a skilled person.Fc binding proteins of the invention may be prepared by any of the manyconventional and well-known techniques such as plain organic syntheticstrategies, solid phase-assisted synthesis techniques or by commerciallyavailable automated synthesizers.

In some embodiments of the first aspect, the multimer is ahomo-multimer, e.g., the amino acid sequences of all Fc binding domainsof the Fc binding protein are identical.

In some embodiments of the first aspect, the multimer is ahetero-multimer, e.g., at least one Fc binding domain has a differentamino acid sequence than the other Fc binding domains within the Fcbinding protein.

In some embodiments of the first aspect, the Fc binding domains aredirectly linked to each other. In other embodiments, the one or more Fcbinding domains are linked to each other with one or more linkers.Preferred in these typical embodiments are peptide linkers. This meansthat the peptide linker is an amino acid sequence that connects a firstFc binding domain with a second Fc binding domain. The peptide linker isconnected to the first Fc binding domain and to the second Fc bindingdomain by a peptide bond between the C-terminal and N-terminal ends ofthe domains, thereby generating a single, linear polypeptide chain. Thelength and composition of a linker may vary between at least one and upto about 30 amino acids. More specifically, a peptide linker has alength of between 1 and 30 amino acids; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 amino acids. It is preferred that the amino acid sequence ofthe peptide linker is stable against caustic conditions and proteases.Linkers should not destabilize the conformation of the domains in the Fcbinding protein. Well-known are linkers comprising small amino acidssuch as glycine and serine. The linkers can be glycine-rich (e.g., morethan 50% of the residues in the linker can be glycine residues). Alsopreferred are linkers that comprise further amino acids. Otherembodiments of the invention comprise linkers consisting of alanine,proline, and serine. Other linkers for the fusion of proteins are knownin the art and can be used.

In some embodiments, the Fc binding protein further comprises anattachment site for covalent attachment to a solid phase (matrix).Preferably, the attachment site is specific to provide a site-specificattachment of the Fc binding protein to the solid phase. Specificattachment sites comprise natural amino acids, such as cysteine orlysine, which enable specific chemical reactions with a reactive groupof the solid phase or a linker between the solid phase and the protein,for example selected from N-hydroxysuccinimide, iodacetamide, maleimide,epoxy, or alkene groups. The attachment site may be directly at the C-or N-terminal end of the Fc binding protein or there may be a linkerbetween the N- or C-terminus and the coupling site, preferably a peptidelinker. In some embodiments of the invention, the Fc binding protein maycomprise a short N- or C-terminal peptide sequence of 3-20 amino acids,preferably 4-10 amino acids, with a terminal cysteine. Amino acids for aC-terminal attachment site may be preferably selected from proline,alanine, and serine, for example, ASPAPSAPSAC (SEQ ID NO: 19), with asingle cysteine at the C-terminal end for coupling. In anotherembodiment, amino acids for a C-terminal attachment site may bepreferably selected from glycine and serine, for example, GGGSC (SEQ IDNO: 22), with a single cysteine at the C-terminal end for coupling.

An advantage of having a C-terminal cysteine is that coupling of the Fcbinding protein can be achieved through reaction of the cysteine thiolwith an electrophilic group on a support resulting in a thioether bridgecoupling. This provides excellent mobility of the coupled protein whichprovides increased binding capacity.

In a second aspect the, present invention is directed to an affinityseparation matrix, comprising an Fc binding protein of the first aspect.

In preferred embodiments of the second aspect, the affinity separationmatrix is a solid support. The affinity separation matrix comprises atleast one Fc binding protein comprising at least one Fc binding domaincomprising any one of SEQ ID NOs: 1-6 or 21.

This matrix comprising the Fc binding protein of the invention is usefulfor separation, for example for chromatographic separation, ofimmunoglobulins and other Fc-containing proteins, such as immunoglobulinvariants comprising the Fc region, fusion proteins comprising a Fcregion of an immunoglobulin, and conjugates comprising a Fc region of animmunoglobulin. An affinity matrix is useful for separation ofimmunoglobulins and should retain the Fc binding property even afterhighly alkaline conditions as applied during cleaning processes. Suchcleaning of matrices is essential for long-term repeated use ofmatrices.

Solid support matrices for affinity chromatography are known in the artand include for example but are not limited to, agarose and stabilizedderivatives of agarose (e.g., Sepharose 6B, PraestoTMPure; CaptivA®,Mabselect®, PROTEIN A Sepharose Fast Flow), cellulose or derivatives ofcellulose, controlled pore glass (e.g., ProSep® vA resin), monolith(e.g., CIM® monoliths), silica, zirconium oxide (e.g., CM Zirconia orCPG®), titanium oxide, or synthetic polymers (e.g., polystyrene such asPoros 50A or Poros MabCapture® A resin, polyvinylether, polyvinylalcohol, polyhydroxyalkyl acrylates, polyhydroxyalkyl methacrylates,polyacrylamides, polymethacrylamides etc) and hydrogels of variouscompositions. In certain embodiments the support comprises a polyhydroxypolymer, such as a polysaccharide. Examples of polysaccharides suitablefor supports include but are not limited to agar, agarose, dextran,starch, cellulose, pullulan, etc, and stabilized variants of these.

The formats for solid support matrices can be of any suitable well-knownkind. Such solid support matrix for coupling the Fc binding protein ofthe invention might comprise, for example, one of the following:columns, capillaries, particles, membranes, filters, monoliths, fibers,pads, gels, slides, plates, cassettes, or any other format commonly usedin chromatography and known to someone skilled in the art.

In one embodiment, the matrix is comprised of substantially sphericalparticles, also known as beads, for example Sepharose or Agarose beads.Suitable particle sizes may be in the diameter range of 5-500 μηη, suchas 10-100 μηη, e.g., 20-80 μηη. Matrices in particle form can be used asa packed bed or in a suspended form including expanded beds.

In an alternative embodiment, the solid support matrix is a membrane,for example a hydrogel membrane. In some embodiments, the affinitypurification involves a membrane as matrix to which the Fc bindingprotein of the first aspect is covalently bound. The solid support canalso be in the form of a membrane in a cartridge.

In some embodiments, the affinity purification involves a chromatographycolumn containing a solid support matrix to which the Fc binding proteinof the first aspect is covalently bound.

The Fc binding protein of the invention may be attached to a suitablesolid support matrix via conventional coupling techniques utilising,e.g., amino-, sulfhydroxy-, and/or carboxy-groups present in the Fcbinding protein of the invention. The coupling may be carried out via anitrogen, oxygen, or sulphur atom of the Fc binding protein. Preferably,amino acids comprised in an N- or C-terminal peptide linker comprisesaid nitrogen, oxygen, or sulphur atom.

The Fc binding proteins may be coupled to the support matrix directly orindirectly via a spacer element to provide an appropriate distancebetween the matrix surface and the Fc binding protein of the inventionwhich improves the availability of the Fc binding protein andfacilitates the chemical coupling of the Fc binding protein of theinvention to the support.

Methods for immobilization of protein ligands to solid supports arewell-known in this field and easily performed by the skilled person inthis field using standard techniques and equipment.

Depending on the Fc binding protein and on the specific conditions, thecoupling may be a multipoint coupling, for example via several lysines,or a single point coupling, for example via cysteine.

In a third aspect, the present invention is directed to the use of theFc binding protein of the first aspect or an affinity matrix of thesecond aspect for affinity purification of immunoglobulins or variantsthereof, i.e. the Fc binding protein of the invention is used foraffinity chromatography. In some embodiments, the Fc binding protein ofthe invention is immobilized onto a solid support as described in thesecond aspect of the invention.

In a fourth aspect the present invention is directed to a method foraffinity purification of a protein comprising an Fc sequence, the methodcomprising:

-   -   (a) providing a solution that contains a protein comprising an        Fc sequence;    -   (b) providing an affinity separation matrix comprising at least        one Fc binding protein of the invention thereto;    -   (c) contacting said affinity separation matrix with the solution        under conditions that permit specific binding of the at least        one Fc binding protein of the invention to a protein comprising        an Fc sequence; and    -   (d) eluting said protein comprising an Fc sequence from said        affinity purification matrix, and    -   (e) optionally comprising washing the affinity matrix between        step (c) and (d).

For the purposes of the disclosed uses and methods, the proteincomprising an Fc sequence is an immunoglobulin molecule or a fragment orderivative thereof that comprises an Fc sequence, consistent with thedefinitions provided herein.

Affinity separation matrixes suitable for the disclosed uses and methodsare those matrixes according to the embodiments described above and asknown to someone skilled in the art.

In some embodiments of the fourth aspect, the elution of theimmunoglobulin from the matrix in step (d) is effected through a changein pH and/or a change in salt concentration. Any suitable solution usedfor elution from Protein A media can be used, for example by a solutionwith pH 5 or lower, or by a solution with pH 1 1 or higher.

In some embodiments, a further step (f) for efficient cleaning theaffinity matrix is added, preferably by using an alkaline liquid, forexample, with pH of 13-14. In certain embodiments, the cleaning liquidcomprises 0.1-1 0.0 M NaOH or KOH, preferably 0.25-0.5 M NaOH or KOH.Due to the high alkaline stability of the Fc binding proteins of theinvention, such strong alkaline solution can be used for cleaningpurposes.

In some embodiments, the affinity matrix can be re-used at least 10times, at least 20 times, at least 30 times, at least 40 times, at least50 times, at least 60 times, at least 70 times, at least 80 times, atleast 90 times, or at least 100 times, due to a repetition of steps (a)to (e), optionally (a) to (f) can be repeated at least 10 times, atleast 20 times, at least 30 times, at least 40 times, at least 50 times,at least 60 times, at least 70 times, at least 80 times, at least 90times, or at least 100 times.

In general, suitable conditions for performing the method of affinitypurification are well known to someone skilled in the art. In someembodiments, the disclosed uses or methods of affinity purificationcomprising the disclosed Fc binding domains may provide elution of atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or at least about 100% of Fc containingproteins at a pH of greater than or equal to 3.5 (e.g., about 4.0, about4.5, about 5.0, about 5.5, about 6.0, or about 6.5). In someembodiments, the elution profile of the disclosed Fc binding domains issuperior to Protein A domain C.

In a fifth aspect, the present invention is directed to a nucleic acidmolecule, preferably an isolated nucleic acid molecule, encoding a Fcbinding protein or Fc binding domain of any embodiment disclosed above.In one embodiment, the present invention is directed to a vectorcomprising the nucleic acid molecule. A vector means any molecule orentity (e.g., nucleic acid, plasmid, bacteriophage or virus) that can beused to transfer protein coding information into a host cell. In oneembodiment, the vector is an expression vector.

In a sixth aspect, the present invention is directed to an expressionsystem which comprises a nucleic acid or a vector as disclosed above,for example a prokaryotic host cell, for example E. coli, or aeukaryotic host, for example yeast Saccharomyces cerevisiae or Pichiapastoris or mammalian cells such as CHO cells.

In a seventh aspect, the present invention is directed to a method forthe production of an Fc binding protein of the first aspect, comprisingthe step(s): (a) culturing the host cell of the sixth aspect undersuitable conditions for the expression of the binding protein in orderto obtain said Fc binding protein; and (b) optionally isolating said Fcbinding protein.

Suitable conditions for culturing a prokaryotic or eukaryotic host arewell-known to the person skilled in the art.

Fc binding molecules of the invention may be prepared by any of the manyconventional and well-known techniques such as plain organic syntheticstrategies, solid phase-assisted synthesis techniques or by commerciallyavailable automated synthesizers. On the other hand, they may also beprepared by conventional recombinant techniques alone or in combinationwith conventional synthetic techniques.

One embodiment of the present invention is directed to a method for thepreparation of an alkaline-stable Fc binding protein comprising at leastone Fc binding domain comprising a sequence of any one of SEQ ID NOs:1-6 or 21, said method comprising the following steps: (a) preparing anucleic acid encoding a Fc binding protein as defined above; (b)introducing said nucleic acid into an expression vector; (c) introducingsaid expression vector into a host cell; (d) cultivating the host cell;(e) subjecting the host cell to culturing conditions under which a Fcbinding protein is expressed, thereby (e) producing a Fc binding proteinas described above; optionally (f) isolating the protein produced instep (e); and (g) optionally conjugating the protein to solid matricesas described above.

In a further embodiment of the present invention the production of theFc binding protein is performed by cell-free in vitrotranscription/translation.

EXAMPLES

The following Examples are provided for further illustration of theinvention. The invention, however, is not limited thereto, and thefollowing Examples merely show the practicability of the invention onthe basis of the above description.

Example 1. Generation of parental Fc binding proteins of the invention

Parental proteins SEQ ID NO: 17 or SEQ ID NO: 18 were initiallygenerated by a shuffling process of naturally occurring Protein Adomains. In more detail, the shuffling process as understood herein isan assembly process resulting in artificial amino acid sequencesstarting from a set of non-identical known amino acid sequences. Theshuffling process comprised the following steps: a) providing sequencesof five naturally occurring Protein A domains E, B, D, A, and C, andProtein A variant domain Z; b) alignment of said sequences; c)statistical fragmentation in silico to identify subsequences that wererecombined, and then d) assembly of new, artificial sequences of thevarious fragments to produce a mosaic product, i.e. a novel amino acidsequence. The fragments generated in step c) were of any length, e.g.,if the fragmented parent sequence had a length of n, the fragments wasof length 1 to n−1.

The relative positions of the amino acids in the mosaic products weremaintained with respect to the starting amino acid sequences. At least90% of positions Q9, Q10, A12, F13, Y14, L17, P20, L22, Q26, R27, F30,131, Q32, S33, L34, K35, D36, D37, P38, S39, S41, L45, E47, A48, K50,L51, Q55, A56, P57 are identical between the artificial amino acidsequences of parental “shuffled” proteins IB24 and IB26, and naturallyoccurring Protein A domains or Protein A domain variants, provided thatposition 4 of IB24 and IB26 is Q. The overall amino acid sequence ofparental proteins IB24 or IB26 is artificial in that it is not more thanabout 85% identical to the overall amino acid sequence of any of thenaturally occurring Protein A domains or domain Z (for example, IB24 orIB26 are only 77% identical to domain B). After the initial artificialproteins was generated, the protein was further modified bysite-specific randomization of the amino acid sequence to further modifythe binding properties. The further modifications were introduced bysite-saturation mutagenesis of individual amino acid residues.

Genes for IB24 and IB26 were synthesized and cloned into an E. coliexpression vector using standard methods known to a skilled person. DNAsequencing was used to verify the correct sequence of insertedfragments.

To generate multimeric Fc binding proteins comprising more than onebinding domain, 2, 3, 4, 5, or 6 Fc binding domains were geneticallyfused.

For specific membrane attachment and purification, a short peptide aminoacid sequence with C-terminal Cys (SEQ ID NO: 19) and optionally astrep-tag (SEQ ID NO: 20) were added to the C-terminus of the Fc bindingproteins.

Example 2. Mutagenesis of Fc Binding Proteins

For site-directed mutagenesis, the Q5® site-directed Mutagenesis Kit(NEB; Cat. No. E0554S) was used according to the manufacturer'sinstructions. A combination of several point mutations was generated byGeneArt™ Strings™ synthesis (Thermo Fisher Scientific). The Strings DNAfragments corresponded to a purified PCR product and were cloned into aderivate of a pET28a vector. Ligation products were transformed into E.coli XL2-blue cells via electroporation. Single colonies were screenedby PCR to identify constructs containing inserts of the right size. DNAsequencing was used to verify the correct sequences.

Example 3. Expression of Fc Binding Proteins

BL21 (DE3) competent cells were transformed with an expression plasmidencoding Fc binding proteins. Cells were spread onto selective agarplates (Kanamycin) and incubated overnight at 37° C. Precultures wereinoculated from single colony in 100 ml 2×YT medium and cultured for 16hours at 37° C. at 160 rpm in a conventional orbital shaker in baffled 1L Erlenmeyer flasks supplemented with 150 μg ml Kanamycin withoutlactose and antifoam. The OD600 readout should be in the range of 6-12.Main culture was inoculated from previous overnight culture with anadjusted start-OD600 of 0.5 in 400 ml superrich medium (modified H15medium 2% Glucose, 5% Yeast extract, 0.89% Glycerol, 0,76% Lactose, 250mM MOPS, 202 mM TRIS, pH 7.4, Antifoam SE15) in 1 L thick-walledErlenmeyer flasks that was supplemented with 150 μg ml Kanamycin.Cultures were transferred to a resonant acoustic mixer (RAMbio) andincubated at 37° C. with 20×g. Aeration was facilitated by Oxy-Pumpstoppers. Recombinant protein expression was induced by metabolizingglucose and subsequently allowing lactose to enter the cells. Atpredefined time points OD600 was measured, samples adjusted to 5/OD600were withdrawn, pelleted and frozen at −20° C. Cells were grownovernight for approx. 24 hours to reach a final OD600 of about 45-60. Tocollect biomass cells were centrifuged at 16000×g for 10 min at 20° C.Pellets were weighed (wet weight) and pH was measured in thesupernatant. Cells were stored at −20° C. before processing.

Example 4: SDS-PAGE Analysis of Expression and Solubility of Fc BindingProteins

Samples taken during fermentation were resuspended in 300 μI extractionbuffer (PBS supplemented with 0.2 mg/ml Lysozyme, 0.5× BugBuster, 7.5 mMMgS04, 40 U Benzonase) and solubilized by agitation in a thermomixer at700 rpm, rt for 15 min. Soluble proteins were separated from insolubleproteins by centrifugation (16000×g, 2 min, rt). Supernatant waswithdrawn (soluble fraction) and the pellet (insoluble fraction) wasresuspended in equivalent amount of urea buffer (8 M urea, 0.2 M Tris, 2mM EDTA, pH 8.5). 50 μI were taken both from the soluble and insolublefraction, and 12 μI 5× sample buffer as well as 5 μI 0.5 M DTT wereadded. Samples were boiled at 95° C. for 5 min. Finally, 8 μI of thosesamples were applied to NuPage Novex 4-12% Bis-Tris SDS gels which wererun in accordance to the manufacturer's recommendations and stained withCoomassie. High level expression of Fc binding proteins was found underoptimized conditions within the chosen period of time (data not shown).All expressed Fc binding proteins were soluble to more than 95%according to SDS-PAGE.

Example 5: Purification of Fc binding proteins

Fc binding proteins were expressed in the soluble fraction of £. co//with a C-terminal StrepTagll (SEQ ID NO: 20). The cells were lysed bytwo freeze/thaw cycles and the purification step was performed withStrep-Tactin®-resin according to the manufacturer's instructions (IBA,Goettingen, Germany). To avoid disulfide formation the buffers weresupplemented with 1 mM DTT.

Alternatively, Fc binding proteins were expressed in the solublefraction of E. coli with a C-terminal StrepTagll. The cells wereresuspended in cell disruption buffer and lysed by a constant celldisruption system (Unit F8B, Holly Farm Business Park) at 1 kbar for twocycles. Purification step was performed with Strep-TactinYesin (IBA,Goettingen, Germany) and additional gel filtration (Superdex 75 16/60;GE Healthcare) using an AKTAxpress system (Ge Healthcare) according tothe manufacturer's instructions. To avoid disulfide formation buffersfor Strep-Tactin-purification were supplemented with 1 mM DTT andcitrate-buffer (20 mM Citrat, 150 mM NaCl, pH 6,0) was used as runningbuffer for gel filtration.

Example 6. The Fc Binding Proteins Bind to IgG with High Affinities (asDetermined with Surface Plasmon Resonance Experiments)

A CMS sensor chip (GE Healthcare) was equilibrated with SPR runningbuffer. Surface-exposed carboxylic groups were activated by passing amixture of EDC and NHS to yield reactive ester groups. 700-1500 RUon-ligand were immobilized on a flow cell, off-ligand was immobilized onanother flow cell. Injection of ethanolamine after ligandimmobilization, ethanolamin and 10 nM Glycin pH 2.0 are injected toremove non-covalently bound Fc binding protein. Upon ligand binding,protein analyte was accumulated on the surface increasing the refractiveindex. This change in the refractive index was measured in real time andplotted as response or resonance units (RU) versus time. The analyteswere applied to the chip in serial dilutions with a suitable flow rate(μI/min). After each run, the chip surface was regenerated withregeneration buffer and equilibrated with running buffer. The controlsamples were applied to the matrix. Regeneration and re-equilibrationwere performed as previously mentioned. Binding studies were carried outby the use of the Biacore® 3000 (GE Healthcare) at 25° C.; dataevaluation was operated via the BIAevaluation 3.0 software, provided bythe manufacturer, by the use of the Langmuir 1:1 model (R1=0). Evaluateddissociation constants (KD) were standardized against off-target. Thebinding affinities of SEQ ID NO: 1 and SEQ ID NO: 2 for human IgGi(Cetuximab), human lgG2 (Panitumomab), and human lgG4 (Natalizumab) areshown in Table 1.

TABLE 1 K_(D) values of Fc binding proteins of the invention SE- Rp- KDvs. KD vs. KD vs. HPLC HPLC IgG 2 IgG 2 IgG 2 CID Ex-change [%] [%] [nM][nM] [nM] Wt C- 100 100 7.2 129 8.0 domain CS24 100 100 17.2 101 12.4CS24 Q9H 100 100 617 6720 403 CS24 D36H 100 100 16.1 169 10.6 CS26 100100 18.4 193 10.9 CS26 Q9H 100 100 668 3320 265 CS26 D36H 100 100 14.4153 11.2

Example 7. Alkaline Stability of Fc Binding Proteins Coupled toSepharose 6B Matrix

Purified Fc binding proteins were coupled to epoxy-activated matrix(Sepharose 6B, GE; Cat. No. 17-0480-01) according to the manufacturer'sinstructions (coupling conditions: pH 9.0 overnight, blocking for 5 hwith ethanolamine). Cetuximab was used as IgGi sample (5 mg; 1 mg/mlmatrix). Cetuximab was applied in saturated amounts to the matrixcomprising immobilized Fc binding protein. The matrix was washed with100 mM glycine buffer, pH 2.5 to elute Cetuximab that was bound to theimmobilized IgG-binding protein. The concentration of the eluted IgG wasmeasured by BLI (quantification with Protein A Octet-sensors andCetuximab as standard) in order to determine the binding activity of theFc binding proteins. Columns were incubated with 0.5 M NaOH for 6 h atroom temperature (22° C.+/−3° C.). The IgG binding activity of theimmobilized proteins was analyzed before and after incubation with 0.5 MNaOH for 6 h. The IgG binding activity of immobilized proteins beforeNaOH treatment was defined as 100%.

FIG. 1 shows that the activity of Fc binding proteins SEQ ID NO: 1 andSEQ ID NO: 2 was higher compared to the activity of the parental proteinIB24 (parental IB26 is comparable to parental IB24; data not shown).Both Fc binding proteins SEQ ID NO: 1 and SEQ ID NO: 2 showed about atleast 30% higher IgG binding activity compared to the parental proteinIB24 after incubation for 6 h at 0.5 M NaOH. Thus, the Fc bindingproteins of the invention show significantly improved stability at highpH, compared to a parental protein.

Example 8. Alkaline Stability of Fc Binding Proteins Coupled toAgarose-Based Chromatography Beads Praesto™ Pure45

Purified Fc binding proteins were coupled to agarose-basedchromatography beads (Praesto™ Pure45, Purolite; Cat. No. PR01262-166)according to the manufacturer's instructions (coupling conditions: pH9.5, 3 hours, 35° C., blocking overnight with ethanolamine). Polyclonalhuman IgG Gammanorm® (Ocatpharm) was used as IgG sample (cone. 2.2mg/ml). Polyclonal hlgG sample was applied in saturated amounts to thematrix comprising immobilized Fc binding protein. The matrix was washedwith 100 mM Citrate buffer, pH 2.0 to elute hlgG that was bound to theimmobilized Fc binding protein. Dynamic binding capacity was determinedby the mass of injected hlgG at 10% breakthrough at 6 min residencetime. Columns were incubated with 0.5 M NaOH for 6 h at room temperature(22° C.+/−3° C.). The IgG binding activity of the immobilized proteinswas analyzed before and after incubation with 0.5 M NaOH for 6 h. TheIgG binding activity of immobilized proteins before NaOH treatment wasdefined as 100%.

FIG. 2 shows that the activity of Fc binding proteins SEQ ID NOs: 1-6 isvery high even after incubation for 6 h at 0.5 M NaOH (at least 87.6%remaining activity for all Fc binding proteins SEQ ID NOs: 1-6 after 6 hincubation at 0.5 M NaOH). All Fc binding proteins of the invention showsignificantly high stability at high pH. FIG. 3 further shows resultsfrom Praesto™ Pure45 matrix and Praesto™ Pure85 matrix.

Example 9. Elution of hlgG from Fc Binding Proteins Coupled toAgarose-Based Chromatography Beads Praesto™ Pure45 and/or Pure85

Purified Fc binding proteins were coupled to agarose-basedchromatography beads (Praesto™ Pure45 or Pure 5) according to themanufacturer's instructions. Polyclonal human IgG Gammanorm® was used asIgG sample (cone. 2.2 mg/ml), loading up to DBC10%. Polyclonal hlgGsample was applied in saturated amounts to the matrix comprisingimmobilized Fc binding protein. In a two-step process, the matrix wasfirst washed with 100 mM Citrate buffer, pH 3.5 and then with 100 mMCitrate buffer, pH 2.0 to elute hlgG that was bound to the immobilizedFc binding protein.

As shown in FIG. 4, greater than 98% of the bound polyclonal human IgGwas eluted at pH 3.5, which was considerably higher than elution fromwild-type Protein A domain C.

1. An Fc binding protein comprising one or more domains, wherein atleast one domain comprises an amino acid sequence having at least 98%sequence identity to any one of SEQ ID NOs: 1-6 and
 21. 2. The Fcbinding protein of claim 1, wherein the protein comprises 2, 3, 4, 5, or6 domains linked to each other.
 3. The Fc binding protein of claim 2,wherein all of the domains comprise sequences selected from any one ofSEQ ID NOs: 1-6 and
 21. 4. The Fc binding protein of claim 2, whereinthe domains are derivatives of SEQ ID NOs 1-6 or 21, and further whereineach derivative has an amino acid sequence that is 100% identical to oneof SEQ ID NOs: 1-6 and 21 except that it has a deletion of 1, 2, or 3amino acids within the first 4 amino acids of its N-terminus and/or adeletion of 1 or 2 amino acids within the first 2 amino acids of itsC-terminus.
 5. The Fc binding protein of claim 2, wherein the protein isa homo-multimer.
 6. The Fc binding protein of claim 2, wherein theprotein is a hetero-multimer.
 7. The Fc binding protein of claim 2,wherein one or more domains are linked to each other directly or withone or more linkers.
 8. The Fc binding protein of claim 7, wherein thelinker is a peptide linker.
 9. The Fc binding protein of claim 1,wherein the protein has less than a 15% reduction in binding capacityfollowing an incubation in 0.5 M NaOH for at least 5 hours.
 10. The Fcbinding protein of claim 1, wherein the Fc binding protein is conjugatedto a solid support.
 11. The Fc binding protein of claim 10, wherein saidFc binding protein further comprises an attachment site forsite-specific covalent coupling of the Fc binding protein to a solidsupport.
 12. An affinity separation matrix comprising an Fc bindingprotein, the Fc binding protein comprising an amino acid sequence havingat least 98% sequence identity to any one of SEQ ID NOS: 1-6 or
 21. 13.The affinity separation matrix of claim 12, wherein the amino acidsequence has at least 98% sequence identity to SEQ ID NO:
 1. 14. Theaffinity separation matrix of claim 12, wherein the amino acid sequencehas at least 98% sequence identity to SEQ ID NO:
 2. 15. The affinityseparation matrix of claim 12, wherein the amino acid sequence has atleast 98% sequence identity to SEQ ID NO:
 3. 16. The affinity separationmatrix of claim 12, wherein the amino acid sequence has at least 98%sequence identity to SEQ ID NO:
 4. 17. The affinity separation matrix ofclaim 12, wherein the amino acid sequence has at least 98% sequenceidentity to SEQ ID NO:
 5. 18. The affinity separation matrix of claim12, wherein the amino acid sequence has at least 98% sequence identityto SEQ ID NO:
 6. 19. The affinity separation matrix of claim 12, whereinthe amino acid sequence has at least 95% sequence identity to SEQ ID NO:21.
 20. A method for affinity purification of a protein comprising an Fcsequence, the method comprising: (a) contacting an affinity separationmatrix comprising at least one Fc binding protein with a solutioncontaining a protein comprising an Fc sequence under conditions thatpermit binding of said at least one Fc binding protein to said proteincomprising an Fc sequence, wherein the at least one Fc binding proteincomprises an amino acid sequence having at least 98% sequence identityto one of SEQ ID NOS: 1-6 or 21; and (b) eluting the bound proteincomprising an Fc sequence from said affinity purification matrix. 21.The method of claim 20, further comprising washing the affinityseparation matrix between steps (a) and (b).
 22. The method of claim 20,wherein there is at least 95% elution of the protein comprising an Fcsequence at a pH of 3.5 or higher.